1,10-decanediyl bismethacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2016 to 05 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay”(22 July 2010)

Deviations:

yes

Remarks:

see "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

see "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,10-decanediyl bismethacrylate

EC Number:

229-745-1

EC Name:

1,10-decanediyl bismethacrylate

Cas Number:

6701-13-9

Molecular formula:

C18H30O4

IUPAC Name:

decane-1,10-diyl bis(2-methylacrylate)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 18.3 – 21.2 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 7 days
Note: In the Preliminary Experiment, mice of 12 weeks of age (22.9-25.0 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.6 – 25.6°C
Relative humidity: 30 - 80 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Food and feeding: Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” produced by ssniff Spezialdiäten GmbH, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water supply: Animals received tap water from the municipal supply from 500 mL bottles, ad libitum.
Bedding: Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).
Identification and randomisation: A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups.
The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.

No. of animals per dose:

Preliminary Irritation/Toxicity Test: 2 animals/dose
Main Test: 4 animals/dose

Details on study design:

Formulation
The solubility of the test item was examined in a short Preliminary Compatibility Test.
The following standard OECD vehicle was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.
The 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) formulations appeared to be solutions by visual examination.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100% (undiluted) and 50% (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored (see “Any other information” for Erythema Scoring table). Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test, no mortality was observed. No test item precipitate was observed on the ears of the animals.
Marked body weight loss (>5% decrease) was detected for both animals in the 100% (undiluted) and 50 % (w/v) groups. The mean body weight loss of these groups was more than 5 %. There were no indications of any irritancy at the site of application.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were slightly enlarged than normal for both animals of the 100% (undiluted) and for one animal of the 50% (w/v) dose groups and they were normal for the other animal of the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).
Taking into account these results, the body weight difference was close to the 5% weight loss in 2 animals of the 100% (undiluted) and in 2 animals of the 50% (w/v) dose group. With the small group sizes in the preliminary test, it is difficult to be certain where the threshold for systemic toxicity exists; hence, to try to be sure of a valid main study and according to the Study Plan, five dose groups will be used in the main experiment so there should be three acceptable dose groups for evaluation. Therefore, 100% (undiluted), 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) doses will be examined in the main test.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

EVALUATION OF THE RESULTS
The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value.
The results were expressed as disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.
The stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The use of the individual approach to calculate the SI made the use of a statistical analysis available.

Interpretation of Results
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values.
Note: In the report, the mean DPM values are shown, but all the data of the individual measurements are kept and archived in the raw data binder.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of intergroup differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of no normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Positive control results:

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 12.0) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Key result

Parameter:

EC3

Value:

17.5

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes in the main study.

PROLIFERATION ASSAY
The appearance of the lymph nodes was larger than normal for all animals of the 100% (undiluted) dose group and for three animals of the 50% (w/v) dose group and in the positive control group. They were slightly enlarged than normal for one animal of the 50% (w/v) dose group and for all animals of the 25% (w/v) dose group, and they were normal for all animals of the 10% (w/v) and 5% (w/v) dose groups and in the negative control group.
The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 5% (w/v) respectively.

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2292 2314 2306 2312	1 2 3 4	Negative (vehicle) control AOO     Mean	21.2 19.6 19.2 18.9 19.7	22.5 20.4 20.2 20.6 20.9	6.1 4.1 5.2 9.0 6.1
2377 2295 2305 2311	5 6 7 8	1,10-decanediyl bis methacrylate 100% (undiluted)     Mean	21.2 19.6 19.2 18.6 19.7	21.0 20.1 20.3 19.9 20.3	-0.9 2.6 5.7 7.0 3.6
2293 2378 2304 2379	9 10 11 12	1,10-decanediyl bis methacrylate 50 (w/v)% in AOO     Mean	21.2 19.2 19.8 18.8 19.8	22.0 21.3 21.4 19.8 21.1	3.8 10.9 8.1 5.3 7.0
2299 2307 2316 2300	13 14 15 16	1,10-decanediyl bis methacrylate 25 (w/v)% in AOO     Mean	21.1 20.6 19.8 18.3 20.0	22.0 20.9 21.2 19.2 20.8	4.3 1.5 7.1 4.9 4.4
2308 2302 2369 2376	17 18 19 20	1,10-decanediyl bis methacrylate 10 (w/v)% in AOO     Mean	20.8 20.0 19.4 19.6 20.0	21.1 20.8 21.0 21.2 21.0	1.4 4.0 8.2 8.2 5.5
2320 2353 2317 2351	21 22 23 24	1,10-decanediyl bis methacrylate 5 (w/v)% in AOO     Mean	20.4 20.3 20.2 18.9 20.0	21.6 20.2 21.8 19.1 20.7	5.9 -0.5 7.9 1.1 3.6
2355 2318 2366 2354	25 26 27 28	Positive control 25 (w/v)% HCA in AOO     Mean	20.5 20.4 19.0 19.4 19.8	21.9 20.7 20.5 19.1 20.6	6.8 1.5 7.9 -1.5 3.7

Notes:

^*: Terminal body weights were measured on Day 6

^#: =(Terminal Body Weights – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Identity Number	Mean DPM^Note	Number of lymph nodes	DPN	Mean DPN	Stimulation Index
Background (5% (w/v) TCA)	-	-	-	-	-	-
Negative control (AOO)	1 2 3 4	724.0 705.5 1552.0 476.5	2 2 2 2	362.0 352.8 776.0 238.3	432.3	1.0
1,10-decanediyl bis methacrylate 100% (undiluted)	5 6 7 8	4443.5 5096.5 5158.5 9531.5	2 2 2 2	2221.8 2548.3 2579.3 4765.8	3028.8	7.0^*
1,10-decanediyl bis methacrylate 50 (w/v)% in AOO	9 10 11 12	1388.0 5409.5 9606.0 6653.0	2 2 2 2	694.0 2704.8 4803.0 3326.5	2882.1	6.7^*
1,10-decanediyl bis methacrylate 25 (w/v)% in AOO	13 14 15 16	3351.5 3426.5 5367.5 3091.5	2 2 2 2	1675.8 1713.3 2683.8 1545.8	1904.6	4.4^*
1,10-decanediyl bis methacrylate 10 (w/v)% in AOO	17 18 19 20	795.0 2279.0 1771.5 797.5	2 2 2 2	397.5 1139.5 885.8 398.8	705.4	1.6^NS
1,10-decanediyl bis methacrylate 5 (w/v)% in AOO	21 22 23 24	2148.5 1953.5 793.0 2637.5	2 2 2 2	1074.3 976.8 396.5 1318.8	941.6	2.2^*
Positive control (25% HCA) in AOO	25 26 27 28	7507.5 9976.5 14103.0 9979.5	2 2 2 2	3753.8 4988.3 7051.5 4989.8	5195.8	12.0^*

Notes:

1. NS = Not Significant (p<0.05, Mann-Whitney U-Test versus negative control)

2. * = Significant (p<0.05, Mann-Whitney U-Test versus negative control)

3. ** = Significant (p<0.01, Mann-Whitney U-Test versus negative control)

4. Note: The background values were in the 32 – 35 range.

 

Summarized Clinical Observations

Group	Animal No.	Identity No.	CLINICAL OBSERVATIONS
			DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (AOO)	2292   2314   2306   2312	1   2   3   4	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 100% (undiluted)	2377   2295   2305   2311	5   6   7   8	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 50 (w/v)% in AOO	2293   2378   2304   2379	9   10   11   12	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 25 (w/v)% in AOO	2299   2307   2316   2300	13   14   15   16	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 10 (w/v)% in AOO	2308   2302   2369   2376	17   18   19   20	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
1,10-decanediyl bis methacrylate 5 (w/v)% in AOO	2320   2353   2317   2351	21   22   23   24	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
Positive control (25% (w/v) HCA in AOO)	2355   2318   2366   2354	25   26   27   28	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free

Note:

1. BT: before treatment; AT: after treatment

 

Results of the Preliminary Irritation / Toxicity Test

 

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2075 2084	1 2	100% (undiluted) 100% (undiluted) Mean	24.5 24.0 24.3	23.2 22.8 23.0	-5.3 -5.0 -5.2
2067 2074	3 4	50% (w/v) 50% (w/v) Mean	25.0 22.9 24.0	23.7 21.2 22.5	-5.2 -7.4 -6.3

Notes:

1.^*: Terminal body weights were measured on Day 6.

2.^#= (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			R	L	R	L	R	L
2075 2084 2067 2074	1 2 3 4	100% (undiluted) 100% (undiluted) 50% (w/v) 50% (w/v)	0.21 0.21 0.21 0.21	0.21 0.22 0.21 0.21	0.23 0.23 0.22 0.22	0.23 0.23 0.21 0.22	0.21 0.21 0.22 0.22	0.22 0.22 0.21 0.22	17.34 16.78 15.67 16.20

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

2. R: Right; L: Left

 

Summarized Clinical Observations

Period	Group	Identity No.	Animal No.	Clinical observations
DAY 1	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 2	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 3	100% (undiluted)	1	2075	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	2084	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	50% (w/v)	3	2067	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	2074	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 4	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0
DAY 5	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0
DAY 6	100% (undiluted)	1	2075	Symptom-free, ES: 0
		2	2084	Symptom-free, ES: 0
	50% (w/v)	3	2067	Symptom-free, ES: 0
		4	2074	Symptom-free, ES: 0

Notes:

1. The clinical observations of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score.

 

 Historical Control Data

 

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

 

CBA/CaOlaHsd mice
	Vehicles
	Acetone : Olive oil 4:1 (AOO)			1% Pluronic PE9200 in water (1% Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	415.2	2922.6	7.5	197.7	1825.3	10.0
Range:  min                max	111.3 847.8	890.3 7674.5	3.3 15.5	23.0 680.8	154.0 6755.8	3.0 33.1
Number of cases	32	32	30	134	134	128

 

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	244.6	2522.6	10.8	488.7	3212.1	7.8
Range:  min                max	140.8 505.8	1201.3 4804.6	6.3 21.3	238.5 934.6	2017.2 4877.5	3.1 14.5
Number of cases	21	21	21	13	13	12

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	235.4	2371.8	10.0	260.2	4888.8	19.5
Range:  min                max	63.3 506.0	817.3 4978.0	6.5 14.4	183.5 383.3	2456.3 8682.5	8.9 36.3
Number of cases	14	14	14	9	10	10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, under the conditions 1,10-decanediyl bis methacrylate was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present 1,10-decanediyl bis methacrylate is 17.5%(w/v).

Executive summary:

The aim of this study was to determine the skin sensitisation potential of 1,10-decanediyl bis methacrylate following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding with the specific requirements of the Sponsor.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (undiluted). The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100% (undiluted) and 50% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 100% (undiluted) was selected as top dose for the main test.

 

In the main assay, twenty eight female CBA/CaOlaHsd mice were allocated to seven groups of four animals each:

- five groups received 1,10-decanediyl bis methacrylate at 100% (undiluted); or diluted in AOO at 50% (w/v), 25% (w/v), 10% (w/v) and 5%(w/v) concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals. No treatment related effects were observed on the mean body weight changes in the main study.

 

The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 5% (w/v) respectively.

 

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present 1,10-decanediyl bis methacrylate was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present 1,10-decanediyl bis methacrylate is 17.5%(w/v).

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1B).